PROTEIN STRUCTURE

Structure of protein
Protein Purification
Size Exclusion Chromatography or Gel
Filtration
PROTEIN MOLECULES
 Protein molecules are biological macromolecules
formed by many amino acids connected by peptide
bonds. Physiologically functional proteins in the body
are all ordered structures.
 Each protein has a certain percentage of amino acid
mass, the sequence of amino acids, and the specific
arrangement position of the peptide chain space.
 Therefore, the protein molecular structure composed
of the sequence of amino acids and the spatial
arrangement of peptide chains is the structural basis
for each protein to have a unique physiological
function.
 Different amino acid sequences and specific spatial
arrangements can create tens of thousands of proteins
in the human body, and complete tens of millions of
physiological functions endowed by life.
 STANDARD
AMINO ACIDS

• sequence of amino acids from N-

terminus to C-terminus is called
the primary structure of the
protein.

• There are 20 kinds of amino acids

that make up human protein, and
the molecular weight of proteins
is relatively large. The sequence
and spatial position of AA in
proteins are almost endless.

• A general formula for the number of

primary structures for a
polypeptide containing n amino
acid units is 20n—e.g. most proteins
contain at least 50 amino acid
residues. [2050 = ~1065].
Determination of Primary Structure

• Separation of polypeptide chain into separate chain.

• Determination of amino acid composition.

• Degradation of protein or polypeptide into smaller fragment.

• Determination of the amino acid sequence.

• Overlapping the peptides to get the order.

• Reverse sequencing technique

Analyse the DNA sequence that codes for protein, then
translate DNA sequence to amino acid sequence.
PROTEIN SEQUENCING
Disulphide Bond Cleavage
Separation of Polypeptide Chain
Disulphide Bond Cleavage
Separation of Polypeptide Chain
Prevent Protein Conformation
 Protease Digestion
* Polypeptides can not be sequence directly if the sequence is longer
than 40 to 100 a.a. Endopeptidases, that cleave at specific sites
within the primary sequence of proteins.

The resultant smaller peptides can be chromatographically separated

and subjected to Edman degradation sequencing reactions.

Trypsin peptide bond C-terminal to R, K, but not if highly specific for positively charged
next to P residues
Chymotrypsin peptide bond C-terminal to F, Y, W but not prefers bulky hydrophobic residues,
if next to P cleaves slowly at N, H, M, L

Elastase peptide bond C-terminal to A, G, S, V, but

not if next to P
Thermolysin peptide bond N-terminal to I, M, F, W, Y, V, prefers small Neutral residues, can
but not if next to P cleave at A, D, H, T

Pepsin peptide bond N-terminal to L, F, W, Y, but exhibits little specificity, requires

when next to P low pH
Endopeptidase V8 peptide bond C-terminal to E
DETERMINATION OF THE AMINO ACID COMPOSITION OF A
POLYPEPTIDE

• Acid Hydrolysis by strong acid (6M HCl) at 110°C for 24 hours.

• Cleaves the peptide bonds and releases the individual amino acids,
which can be separated by cation-exchange chromatography or HPLC.

• The separated amino acids quantitated by heating them with

Ninhydrin—a reagent that forms a purple compound with most amino
acids, ammonia, and amines.

• The amount of each amino acid is determined by spectrophotometrically

by measuring the amount of light absorbed by the Ninhydrin derivative.
ACID HYDROLYSIS:

It degrades serine, threonine, tyrosine, tryptophan.

ALKALINE HYDROLYSIS:

A Purified sample is hydrolysed with a strong base e.g: NaOH

It does not destroy tyrosine , tryptophan and glutamine but it destroys

Serine, threonine, arginine.

ENZYMATIC HYDROLYSIS:

To cleave the proteins into a small number of peptide fragments.

Cyanogen Bromide (CNBr) splits polypeptide chain only on the carboxylic side
of methionine residues.
 SEQUENCING OF PROTEIN
End group analysis

• Identification of N- terminal and C- terminal amino

acids in a polypeptide chain is called End group
analysis.

• Identification of N- terminal
• 1) Sanger’s Method
• 2) Edman’s Degradation Technique
• 3) Dansyl Chloride Method
N-TERMINUS IDENTIFICATION
 (a) Sanger's Reagent: This sequencing technique
utilizes the compound, 2,4-dinitrofluorobenzene
(DNF) which reacts with the N-terminal residue
under alkaline conditions. The derivatized amino
acid can be hydrolyzed and will be labeled with a
dinitrobenzene group that imparts a yellow color
to the amino acid. Separation of the modified
amino acids (DNP-derivative) by electrophoresis
and comparison with the migration of DNP-
derivative standards allows for the identification
of the N-terminal amino acid.
EDMAN DEGRADATION
 can label and analyze the N-
terminal amino acid
sequence without
disturbing the peptide
bond.

 But this method is not

suitable for N-terminal
blocking or chemical
modification.
 Protein C-terminus Identification
No reliable chemical techniques exist for sequencing the C-
terminal amino acid of peptides
Enzymatic digestion
eg. Exopeptidases such as carboxypeptidase cleaves a C-terminal
residue from a polypeptide. The cleaved product is analyze by HPLC
chromatography.
Disadvantages: the reaction rate varied at cutting the C-terminal residue.

Carboxypeptidase Will not cleave when C- Carboxypeptidase B Will not cleave when
A terminal residue = R, K or P C- terminal residue
or if P resides next to = R or K or when P
terminal residue resides next to
terminal reside

Carboxypeptidase All free C-terminal residues, Carboxypeptidase Y All free C-

C pH optimum = 3.5 terminal
residues, slowly
at G residues
 Chemical Digestion of proteins
(a) Cyanogen bromide (CNBr) causes specific cleavage at the C-terminal side of
Met residues. The number of peptide fragments that result from CNBr
cleavage is equivalent to one more than the number of Met residues in a
protein.

b) hydrazinolysis (NH2-NH2) yield aminoacyl hydrazides except for the C-

terminal residue in free amino acid form. Analyzed by chromatography.
Disadvantage: Induced side reaction. Thus, limited application to
carboxypeptidase- resistant polypeptides.
AKABORI METHOD
ENZYMATIC METHOD FOR PROTEIN N-TERMINUS
IDENTIFICATION
 Reconstructing the Sequence

Compare cleavage by trypsin and staphylococcal protease on a

typical peptide:
• Trypsin cleavage:
A-E-F-S-G-I-T-P-K L-V-G-K

• Staphylococcal protease: F-S-G-I-T-P-K L-V-G-K-A-E

• The correct overlap of fragments:

L-V-G-K A-E-F-S-G-I-T-P-K
L-V-G-K-A-EF-S-G-I-T-P-K
• Correct sequence:
L-V-G-K-A-E-F-S-G-I-T-P-K
AUTOMATIC PROTEIN SEQUENCE ANALYSIS
Protein samples can be blotted directly onto PVDF from
polyacrylamide gels using standard techniques.

Digested amino acids are derivatized by dansyl chloride, Edman's

reagent, or (OPA)o-phthalaldehyde + 2-mercaptoethanol.

At each cycle the derivatized amino acid is transferred to the

HPLC for analysis and quantification.

In most cases, 10 picomoles are required for sequencing.

It is possible to obtain sequence data on as little as 1-2 picomoles

of clean peptide or 5 picomoles of clean protein.
EXAMPLES OF IMPORTANT PROTEINS FOR
PPI
Structural Levels of Organization
 Membrane Receptors
They are integral structure and glycoprotein in nature. Mol. Wt. about 140
to 150 Kda.

1. Ligand binding part

2. Hydrophobic structure crossing membrane
3. Cytoplasmic part helps in signal transduction

Types of receptor
Catalytic receptor – Associated with enzymatic activity. Tyrosine specific
protein kinase.
Example: Insulin and Growth Factor Receptors.

Receptors containing ion channels

Examples: Nicotinic acetylcholine receptor of muscles and nerves; contain
Na+ channel, Glutamate receptor, GABA & Glycine receptor

Receptor containing intracellular messenger molecule

Beta adrenergic receptor, Adenylate cyclase system, G protein, Calcium/
Phosphotidyl Inositol system, Diacylglycerol, IP3 etc.
SIGNALLING MOLECULES

 Mostsignalling molecules
cannot pass through the cell
membrane
 Their receptors are in the cell
membrane
 Small hydrophobic signal
molecules can diffuse
directly into the cell
cytoplasm
 Their receptors are cytoplasmic
or nuclear
SIGNALLING MOLECULES

 Hormones
 Insulin,
 Cortisol
 etc
 Local mediators
 Epidermal Growth Factor (EGF),
 Platelet Derived Growth Factor (PDGF)
 Fibroblast Growth Factor (FGF)
 TGF
 Cytokines, e.g. Interferons, Tumour necrosis factor (TNF)
RECEPTORS
 There are three main classes of receptors….
 Ion-channel-linked receptors

 G-protein-linked receptors

 Enzyme-linked receptors
RECEPTORS

 Ion channel-linked
receptors are important in
neural signalling
 G-protein and enzyme
linked receptors respond by
activating cascades of
intracellular signals
 These signals alter the
behaviour of the cell
G-PROTEIN-LINKED RECEPTORS

 G-protein-linked receptors activate a class of GTP-

binding proteins (G-proteins)
 G proteins are molecular switches

 They are turned on for brief periods while bound to

GTP
 They switch themselves off by hydrolysing GTP to GDP
 G PROTEINS
 Some G proteins directly regulate ion channels
 Others activate adenylate cyclase, thus increasing
intracellular cyclic AMP
 Some activate the enzyme Phospholipase C, thus
increasing intracellular inositol triphosphate (IP3)
and Diacylglycerol (DAG)
ENZYME-LINKED RECEPTORS
 Many receptors have intracellular domains with
enzyme function
 Most are receptor tyrosine-kinases
 They phosphorylate tyrosine residues in selected
intracellular proteins
 These receptors are activated by growth factors,
thus being important in cell proliferation
RECEPTOR TYROSINE KINASES

 Receptor tyrosine kinase activation results in

assembly of an intracellular signalling complex
 This complex activates a small GTP-binding
protein, Ras
 Ras activates a cascade of protein kinases that
relay the signal to the nucleus
 Mutations that make Ras hyperactive are a
common way of inducing increased proliferation
in cancer
 INSULIN RECEPTOR

The insulin receptor (IR) is a transmembrane receptor that

is activated by insulin, IGF-I, IGF-II and belongs to the large
class of receptor tyrosine kinase

It regulates glucose metabolism.

It is encoded by INSR gene. It is homodimer or heterodimer

320 Kda disulphide linked transmembrane insulin receptor.

Insulin….Receptor......Autophosphorylation…..Insulin
receptor substrate (IRS 1)…….phosphoinositide 3-kinase
(PIK3)………phosphatidylinositol 4,5 biphosphate to
PIP3…..PIP3…..Protein kinase B……translocation of
glucose transporter 4 (GLUT4)
 EPIDERMAL GROWTH FACTOR RECEPTOR

The epidermal growth factor receptor (EGFR; ErbB-

1; HER1 in humans) is a transmembrane protein that is
a receptor for members of the epidermal growth factor family
(EGF family) of extracellular protein ligands

The epidermal growth factor receptor is a member of

the ErbB family of receptors, a subfamily of four closely
related receptor tyrosine kinases: EGFR (ErbB-
1), HER2/neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-
4)

Deficient signaling of the EGFR and other receptor tyrosine

kinases in humans is associated with diseases such as
Alzheimer's, while over-expression is associated with the
development of a wide variety of tumors.
 PLATELET DERIVED GROWTH FACTOR RECEPTOR
Platelet-Derived Growth Factors (PDGFs) are a family of four
cystine-knot-type growth factors (PDGF-A, -B, -C and -D)
which control the growth of connective tissue cells such as
fibroblasts and smooth muscle cells

There are two types of receptors for PDGFs, PDGFRα and

PDGFRβ, which belong to the class III receptor tyrosine
kinases (RTKs), and have different expression patterns and
physiological roles.

PDGFRα signaling controls gastrulation and the development

of several organs such as lung, intestine, skin, testis, kidney,
bones, and neuroprotective tissues.

PDGFRβ signaling is better recognized as an essential

regulator of early hematopoiesis and blood vessel formation
SIGNALLING PATHWAY INTERACTIONS

 There are many signalling molecules and receptors

 A given cell expresses only a subset of receptors
 Different intracellular signalling pathways interact
 This enables cells to respond appropriately to complex
combinations of signals
 Microbial Rhodophsin or Bacterial Rhodophsin

Microbial rhodopsins or bacterial rhodopsins are retinal-binding

proteins that provide light-dependent ion transport and sensory functions
in halophilic and other bacteria.

They are integral membrane proteins with seven transmembrane

helices.
This protein family includes light-driven proton pumps, ion
pumps and ion channels, as well as light sensors.

Proteins
from halobacteria include bacteriorhodopsin and archaerhodopsin,
which are light-driven proton pumps

The term bacterial rhodopsin originally referred to the first

microbial rhodopsin discovered, known as bacteriorhodopsin archaeal
origin, from Halobacterium salinarum
The Ion-translocating Microbial Rhodopsin (MR)
Family catalyze light-driven ion translocation across
microbial cytoplasmic membranes or serve as light
receptors. Most proteins of the MR family are all of about
the same size (250-350 amino acyl residues) and possess
seven transmembrane helical spanners with their N-
termini on the outside and their C-termini on the inside.
There are 9 subfamilies in the MR family.

Bacteriorhodopsins pump protons out of the cell

Bacteriorhodopsin is a light-driven H+ ion transporter found

in some haloarchaea, mostly in Halobacterium salinarum.
The proton-motive force generated by the protein is used
by ATP synthase to generate adenosine triphosphate (ATP)
Bacteriorhodopsin is a 27 kDa integral membrane
protein usually found in two-dimensional crystalline
patches known as "purple membrane", which can occupy
almost 50% of the surface area of the archaeal cell. Each
monomer has seven transmembrane alpha helices and
an extracellular-facing, two-stranded beta sheet.

Bacteriorhodopsin molecule is purple and is most efficient

at absorbing green light (in the wavelength range 500-
650 nm).
Bacteriorhodopsin is a light-driven proton pump. It is the retinal
molecule that changes its isomerization state from all-trans to 13-
cis when it absorbs a photon. The surrounding protein responds to
the change in the chromophore shape, by undergoing an ordered
sequence of conformational changes (collectively known as the
photocycle). Amino acids in the core of the protein, including Asp85,
Asp96 and the Schiff base N atom (Lys216). These sequential
changes result in the transfer of one proton from the intracellular
side to the extracellular side of the membrane for each photon
absorbed by the chromophore.